sacmicro.files.wordpress.com€¦  · Web viewThe quantity of the growth factor in a test sample is determined by comparing the extent of growth caused by the unknown sample with

Unit-6: Nutritional Requirements, Culture Media, Culture Methods and Pure Cultures

Nutritional requirements

Microorganism also needs nutrition for their growth, synthesis of cell material and multiplication. Microorganisms draw nutrients from the environment in which they preset. Those substances that supply requirements for their growth and energy are called nutrients. Most of these nutrients are in the form of suspension of particulate matter or aqueous solution in sea, river, lakes, ground water, animal blood, lymph, sewage or other decomposing organic matter.

The physical and the chemical nature of the habitat determine the kind of organisms it will support. Nutritive and environmental requirements of microbes vary widely.

Along with water, seven elements –carbon, oxygen, nitrogen, hydrogen, phosphorus, sulfur and potassium are the major components of all living organisms. They constitute 50%, 20%, 14%, 8%,, 3%,and 1% respectively in the order of decreasing abundance. These are called Macronutrients. In addition to these, microorganisms also need certain elements in minor amounts. These are also important for normal cell function. These are called Micronutrients. Certain growth factors like organic compounds-amino acids, purines, pyrimidines and vitamins are also required for the organisms.

Table-1- Macro Elements

Element Form of Nutrient Found in the Environment

Carbon( C) Co2, Organic compounds

Hydrogen( H ) H2O, Organic Compounds

Oxygen ( O ) H2O, Organic Compounds, O2

Nitrogen ( N) NH3,NO3¯ , N2, Organic nitrogen compounds

Phosphorus(P) PO43-

Sulfur (S) H2S, SO42- ,organic Sulfur compounds , metallic

sulfides

Potassium(K) K+ in solution or as various K salts

Magnesium( Mg) Mg+ in solution or as various Mg salts

Sodium (Na) Na+ in solution or as Nacl or other Na salts

Calcium ( Ca) Ca+ in solution or as CaSO4 or other Ca salts

Iron (Fe) Fe2+, or Fe3+ in solution or as FeS, Fe( OH )3,or many other Fe salts

1

Table-2 – Examples of MicroelementsElement Cellular Functions

Cobalt( Co ) Vitamin B-12; transcarboxylaseCopper (Cu) Respiration, cytochrome,c Oxidase,photosynthesis,

plastocyanin,Manganese(Mn) Activator of many enzymes

Molybdenum(Mb) Certain flavin containing enzymes, Nitrtae reductase

Nickel (Ni) Most hydrogenases,CoenzymeF430 of methanogenase

Selenium (Se) Formate dehydrogenase;somehydrogenaseZinc(Zn) Carbomic anhydrase, alcohol dehydrogenase

Requirement for Oxygen: All organisms require oxygen for cell components. Oxygen is provided in various forms such as water and organic compounds and molecular oxygen.Sulfur Requirement: Sulfur is obtained in a reduced form as H2S or other sulfide or sulfur containing organic compounds. The sulfur requirements can be met by organic nutrients such as amino acids and peptone.Requirement for Nitrogen: This must be in reduced form (ex: Nitrogen as in amino acids). Certain species of true fungi, most blue-green algae and a few genera of bacteria can enzymatically reduce atmospheric nitrogen for of organic compounds in the cell, a process called biological nitrogen fixation.Requirement for Phosphorus: The non –metallic element, phosphorus cab be used as a nutrient when provided in inorganic form as phosphate salts ( KH2PO4).It is present in the 3% dry weight of cell.Requirement for Carbon: Organisms that perform photosynthesis and bacteria that obtain energy from the oxidation of inorganic compounds contain oxidized forms of carbon and CO2 as the sole source of cellular carbon (autotrophs).All other organisms obtain carbon largely from organic substances

Table-3 -Examples of Growth factors: Vitamins& their functionsVitamin Function

Folic acid One carbon metabolism; methyl group transfer

Nicotinic acid(niacin) Precursor of NAD; electron transfer in red-ox reactions

2

(heterotrophs). Organic substances have a double nutritional role. They serve as a source of carbon and as a source of energy. Many microorganisms can use a single organic compound to supply both these nutritional requirements while others need a variable number of additional organic compounds as nutrients.

Microorganisms are very diverse with respect to the utilization of various kinds of organic compounds for their carbon and energy requirement. A few groups of organisms are specialized to use a restricted source of carbon for their requirement. Ex: Methane oxidizing bacteria can use only methane and methanol as a carbon source. Most organisms that depend on organic carbon source also require CO2 as nutrient in very small amounts because this compound is utilized in a few biosynthetic reactions. Few bacteria and fungi require a relatively high concentration of CO2 in the atmosphere for satisfactory growth in organic media.Saprophytes are heterotrophic organisms which obtain organic compounds from waste and dead remains and serve as scavengers in environment. They play an important role in decomposing organic matter.Parasites are also heterotrophs that live inside other living cell or tissues and cause damage to the host organism. Obligate parasite is totally adapted to parasitic existence. Ex: Viruses, Rickettsias.Growth FactorsMicroorganisms often grow and reproduce when minerals and sources of energy, carbon, nitrogen, phosphorus, and sulfur are supplied. These organisms have the enzymes and pathways .Microbial Nutrition essary to synthesize all cell components required for their wellbeing. Many microorganisms, on the other hand, lack one or more essential enzymes. Therefore they cannot manufacture all indispensable constituents but must obtain them or their precursors from the environment. Organic compounds required because they are essential cell components or precursors of such components and cannot be synthesized by the organism are called growth factors. There are three major classes of growth factors: (1) amino acids, (2) purines and pyrimidines, and (3) vitamins. Amino acids are needed for protein synthesis, purines and pyrimidines for nucleic acid synthesis. Vitamins are small organic molecules that usually make up all or part of enzyme cofactors (see section 8.6), and only very small amounts sustain growth. The functions of selected vitamins, and examples of microorganisms requiring them, are given in table 5.3. Some microorganisms require many vitamins; for example, Enterococcus faecalis needs eight different vitamins for growth. Other growth factors are also seen; heme (from hemoglobin or cytochromes) is required by Haemophilus influenzae, and some mycoplasmas need cholesterol. Knowledge of the specific growth factor requirements of many microorganisms makes possible quantitative growthresponse assays for a variety of substances. For example, species from the bacterial genera Lactobacillus and Streptococcus can be used in microbiological assays of most vitamins and amino acids. The appropriate bacterium is grown in a series of culture vessels, each containing medium with an excess amount of all required components except the growth factor to be assayed. A different amount of growth factor is added to each vessel. The standard curve is prepared by plotting the growth factor quantity or concentration against the total extent of bacterial growth. Ideally the amount of growth resulting is directly proportional to the quantity of growth factor present; if the growth factor concentration doubles, the final extent of bacterial growth doubles. The quantity of the growth factor in a test sample is determined by comparing the extent of growth caused by the unknown sample with that resulting from the standards. Microbiological assays are specific, sensitive, and simple. They still are used in the assay of substances ike vitamin B12 and biotin, despite advances in chemical assay techniques. The observation that many microorganisms can synthesize large quantities of vitamins has led to their use in industry. Several water-soluble and fat-soluble vitamins are produced partly or completely using industrial fermentations. Good examples of such vitamins and the microorganisms that synthesize them are riboflavin (Clostridium, Candida, Ashbya, Eremothecium), coenzyme A (Brevibacterium), vitamin B12 (Streptomyces, Propionibacterium,

3

Nutritional ClassificationNutritional classifications are based on the two important parameters. a) energy source and b) carbon source.Organisms that use light as an energy source are called Phototrophs. Organisms that are dependent on a chemical energy source are termed as Chemotrophs.Organisms able to use CO2 as a principal carbon source are termed autotrophs. Organisms dependent on an organic carbon source are termed heterotrophs. By means of these criteria 4 major nutritional categories can be distinguished.

Table No: 4 Nutritional ClassificationsClassification Energy Source Carbon Source1)Phototrophs( Photolithotroph)Ex: Plants, Algae, Photosynthetic bacteria

Light Carbon dioxide

2) photoheterotrophs(Photoorganotrophs)Ex: Purple bacteria, Green bacteria

Light Organic compound

3) Chemotrophs( Chemolithotrophs)Ex: Iron bacteria

Reduced inorganic compounds

NH4, NO2, N2

Organic compound

4) Photoheterotrophs(Chemoorganotrophs)Ex: Most bacteria, Fungi Protozoa.

Organic compound Organic compound

This system of classification is a rather arbitrary as many phototrophic organisms can also grow as Chemotrophs. Ex: Pseudomonas pseudoflava can use either the organic compound glucose orthe inorganic compound H2 as its source of electrons.Obligate Phototrophs are strictly dependent on light for its energy source and on CO2 for its principal carbon source but facultative Phototrophs are not dependent.Prototrophs are those organisms that can derive all carbon requirements from the principal carbon source.Auxotrophs organisms are those that require one or more organic nutrients (growth factor) along with the principal carbon source. Both Prototrophs and auxotrophs can occur among the organisms assigned to any one of the four major nutritional categories.

Physical (Environmental) Conditions Required for GrowthMicroorganisms exhibit diverse response to the physical conditions in their habitat. Some of the important physical factors that affect growth are: a) temperature, b)pH, c) Gases, d)water, e ) solutes f) osmotic pressure and g) hydrostatic pressure h) radiation.a) Temperature: Temperature determines the rate of growth, the total amount of growth, the metabolic activities and morphology of organisms. Each group microorganism can grow only within a growth

4

temperature range characteristic of the species. The temperature relationship of the organisms are usually descried by the 3 cardinal temperaturesThe lowest temperature at which organisms grow is the minimum growth temperature. Most organisms will survive for a varying length of time below this temperature but will show negligible growth.The maximum growth temperature is the highest temperature at which growth occurs. A temperature slightly above this point frequently kills microorganisms.The optimum growth temperature is defined as the temperature at which most rapid rate of growth (multiplication) occurs. For most organisms, the optimum growth occurs over a temperature range rather than at a fixed temperature. Generally the upper limit of optimum temperature is a few degrees below the maximum temperature.

Table-No:5-Classification of Bacteria according to Growth temperatureGroup Temperature Range Sub-Division

PsychrophilesEx: Polaromonas vaculata

Grow well at OºCa) Can’t grow at temp. >19-22ºC.b ) May grow between 30-35ºC

Obligate PsychrophileFacultative Psychrophile

MesophilesEx: Escherichia coli

Optimum growth at temp. less than 45ºCa) Opt. growth temp. 20-35ºCb) Opt. growth temp. 35-45ºC

SaprophytesAnimal parasites

ThermophilesEx: Bacillus stearothermophilus

Optimum growth temperature is above 45ºCa) growth above 50ºCb) growth at 37ºC and above 50ºC

Obligate thermophileFacultative thermophile

Organisms that grow at extremely hot habitats such as hot springs, geysers and deep sea hydrothermal vents are called hyperthermophiles.Ex: Pyrolobus fumari. Organisms that withstand heat but do not grow at temperature are called thermoduric.

b) pH: For most bacteria the optimum pH for growth lies between 6.7—7.5. Orgainisms that grow between pH 5.5 and 8.0 are called neutrophiles. Ex: Escherichia coliHowever, a few bacteria prefer more extreme pH for growth. Organisms that grow best at acidic pH are called acidophiles.

Ex: Thiobacillus thioxidans which oxidizes Sulfur to sulfuric acid can tolerate a pH upto 0.5-5; optimum pH for growth being 2 -3.5. A few have very high pH optima for growth. These are called alkaliphiles.Ex: Bacillus species; B.firmis.Bacteria that infect human urinary tract and hydrolyze urea to free ammonia can grow at pH 11. Some extremely alkaliphilic bacteria are also halophilic (salt –loving) Ex: Archaea bacteria. They are used in house hold detergents manufacturing industries as they produce hydrolytic and lipase enzymes which function well at alkaline pH.Yeast prefer acidic medium while molds also prefer acidic medium.When organisms are inoculated in a medium originally adjusted to a given pH, it is likely to change the pH depending upon the type of microbial activity and composition of the medium.

Radical shifts in pH can be prevented by incorporating a buffer-a composition of KH2PO4 and K2HPO4 is widely employed in bacteriological media.MgCO3 and CaCO3 are also used to prevent a drop in pH as acid is produced. Phosphates are used widely in preparation of media because they are the only inorganic agents that buffer in physiologically important range around neutrality and are non-toxic to microorganisms. Generally

5

about 5 grams of potassium phosphates per liter of medium can be tolerated by bacteria and fungi. Carbonates are used when a great deal of acid is produced. Insoluble carbonates are also used as it does not produce strong alkaline condition. In addition, acid –forming colonies dissolve the precipitated chalk and become surrounded by clear zones. They can be easily recognized against the opaque background of the medium.c).Gaseous Requirements: i) Most microorganisms require oxygen for respiration. In them, oxygen serve as the terminal electron acceptor and such organisms are called obligate aerobes. Ex: Nitrobacter.Some organisms do not use oxygen as the terminal electron acceptor though oxygen is a component of their cellular material. They are called obligate anaerobes.Ex: ClostridiaSome organisms are micro aerophilic as they require only a little oxygen.Ex: LactobacillusFacultative anaerobes can grow either in the presence or absence of oxygen.Ex: Escherichia coli.ii) Carbon dioxide requirement: All microorganisms utilize CO2 for growth. Autotrophs need sufficient amounts of CO2. In case of autotrophs that grow in an anaerobic environment, Co2 can met by providing buffer such as CaCO3 or NaCO3 which release CO2. Many parasitic bacteria that occur in blood, tissues and gastrointestinal tract are adapted to a higher CO2 content in their habitat than in air. Such bacteria need 10% CO2. d) Water: Microorganisms vary widely in their requirement for water. Water availability is generally expressed in terms such as water activity. ( aw). It is ratio of the vapor pressure of the air in equilibrium with a substance or solution to the vapor pressure of pure water. The values vary between 0 and 1. Microorganisms can grow over a range of water activities of 0.98 – 0.6. Most of bacteria need water activities ( aw) of more than 0.98 except halophilic bacteria with a aw of 0.75.Yeast needs aw of 0.88 and molds can grow at aw of 0.8e) Solutes: Many microorganisms keep the osmotic concentration of their protoplasm somewhat above that of the habitat by the use of compatible solutes’ so that the plasma membrane is always pressed firmly against their cell wall. Compatible solutes are solutes that are compatible with metabolism and growth when at high intracellular concentrations. These are highly water soluble sugars, sugar alcohol, other alcohols, amino acids or their derivates. Compatible solutes are either synthesized by microorganism directly or in some cases accumulated from the environment.f) Osmotic pressure: The stability and behavior of enzymes in cells are greatly influenced by the ionic strength exerted in the form of osmotic pressure. A high concentration of either sugar or salt is lethal to many organisms. Microorganisms have the ability to grow at different osmotic pressures.Osmotolerants are those which grow over wide range osmotic concentrations. Ex: Staphylococcus aureus Yeast and molds have the ability to grow in the habitats of a high concentration of sugar and salt. Halophiles grow in presence of NaCl or other salts at a concentration above 0.2 M Ex: Halobacterium and extreme halophiles are adapted to grow at high concentrations of salt of about 2M. Marine bacteria require high concentration of sodium chloride for their optimum growth.g) Hydrostatic pressure: Hydrostatic pressure exerted by a water column as result of the weight of the column can influence microbial growth rates. Each 10 meter of water depth is equivalent to approximately 1 atmosphere. Most microorganisms are relatively tolerant to the hydrostatic pressure in most natural systems but can not withstand the extremely high hydrostatic pressure that characterizes deep ocean regions. Hydrostatic pressure more than 200 atmospheres generally inactivate enzymes and disrupt membrane transport process. However some microorganisms referred as barotolerant which survive and adapt high hydrostatic pressure. Barophilc are that grow more rapidly at high pressure. Ex: Photobacterium.

6

h) Radiation: Sunlight is the major source of radiation on the earth. Visible light is immensely beneficial as it is the source of energy for photosynthesis. Photosynthetic organisms possess pigments like chlorophyll, bactriochlorophyll, cytochromes and flavins which can absorb light energy and covert it into chemical energy.

Culture MediaMuch of the microbiology depends on the ability to grow and maintain microorganisms in the laboratory and this is made possible with availability of culture media. A culture medium is a solid or liquid preparation used to grow, transport and store microorganisms. A good culture medium contains all nutrients essential for growth of microorganism. The exact composition of a medium depends upon the species that is being cultured. Knowledge of a microorganism’s normal habitat is useful in selecting an appropriate culture medium.Composition of culture media: i) Water: Sources of hydrogen and oxygen, ii) Electrolyte-sodium chloride, iii) Peptone: It is a complex mixture of partially digested proteins from animal or vegetable source. Commercial peptones contain peptone, proteases, polypeptides, amino acids and inorganic salts including phosphates, minerals and accessory growth factors like riboflavin. iv) Meat extract and yeast extract: These contain protein degradation product s, carbohydrates, inorganic salts and certain growth factors. v) Blood: It enriches media, usually, 5-10% defibrinated horse or sheep blood and sometimes serum. vi) Agar: It is derived from sea algae (Geledium species), contains carbohydrates, a small amounts of protein –like material, traces of long chain fatty acids and a variety of impurities. It is usually used in proportion of 2-3% as a solidifying agent of culture medium.Types of Media: Different types of culture media are being used based on their purpose, function and application. Frequently a medium is used particularly to select, grow specific microorganism or to help identify a particular species.Culture media based on consistency, may be I. a) Liquid, b) solid and c) semi solid.

7

II. Synthetic or Defined Medium: A medium in which all components are known. It contains a source of carbon, nitrate or ammonium salt, sulfate, phosphate and a variety of minerals.Ex: Defined media for Escherichia coli; composition g /liter: Glucose-1 g, Na2HPO4-16.4 gK2HPO4 - 1.5g (NH4)2SO4-2 g MgSO4.7H2O-200mg CaCl2-10.0g FeSO4.7H2O -0.5Mg Final pH-6.8 -7.

III. Complex media: Media that contain ingredients of unknown chemical composition.Nutrient broth: Composition g /liter:Beef extract-3 gPeptone-5gWater-1000mlNutrient Agar: Beef extract-3 gpH-7.3Nutrient Agar: It is prepared by adding 2-3% agar to the nutrient broth.Peptone-5gAgar-15gWater-1000mlpH-7.3

Nutrient broth and nutrient agar are also called basal media and they are routinely used in microbiology laboratories.Special media: When certain ingredients are added to a basal medium for growth of microorganisms, it is called special media (Complex medium). Virtually all special media are complex medium.IV. Enriched Medium: When some special nutrients such as blood or serum, egg or meat pieces are added to basal media, it is converted into enriched media. Ex: Blood agar, Loeffler’s serum media. These media are used to culture nutritionally exacting (fastidious) organisms. Ex: Streptococci.V. Enrichment Medium: It is a liquid medium enriched by incorporation of special substances that favors a particular organism or inhibits growth of competitors. Ex: Selenite broth.VI. Selective Medium: These media provide nutrients that enhance the growth and predominance of particular type of bacterium and do not enhance other types of organisms that may be present. Ex:Bile salt agar for isolation cholera bacteria.VII. Differential Medium: When culture medium containing certain substances helps to distinguish different kinds of bacteria, then it is called differential media. Ex:MacConkey’s agar- lactose fermenters form pink colonies while non lactose fermenters form colorless or pale colonies.VIII. Indicator Medium: When certain indicator (neutral red) or reducing substances (potassium tellurite) is incorporated, it is called indicator medium. The color of the medium changes with the growth of bacteria. Ex: Salmonella typhi grow as black metallic sheen colonies on Wilson and Blair medium.IX. Transport Medium: These media are used to transport and maintain organisms in viable condition in specimen collected from far off places of laboratory. Ex: Stuart’s medium for Gonococci.X. Assay Medium: Media used in assay of vitamins, amino acids and antibiotics.XI. Media for Characterization: A wide variety of media are being used to determine the type of growth and to determine their ability to produce certain chemical changes .Ex:Simmon’s Citrate medium- Media used in biochemical tests.XII. Maintenance Media: Medium used to maintain culture for long periods; usually such media do not contain glucose to prevent rapid growth and death of organisms.XIII. Anaerobic Culture Media: These are used to culture anaerobic bacteria. Ex: Robertson’s cooked meat medium.IVX. Dehydrated Media: Commercially available ready to use media which can be reconstituted by dissolving required amounts in known volumes of distilled water.Culture Methods and Pure CulturesThe culture of organisms is required for the following purposes in microbiology:a) to isolate bacteria in pure cultureb) to demonstrate their propertiesc) to obtain sufficient growth for preparation of antigens and for other tests

8

d) to determine sensitivity to antibioticse) to estimate viable countsf) to maintain stock culture.Cultivation of Aerobic Bacteria: In order to grow aerobic or facultative bacteria, the culture media in tubes or small flaks are inoculated, incubated under normal atmosphere conditions. In order to culture in larger quantities, medium is exposed to the atmosphere by dispensing the medium in shallow layers for which special containers are available. Aeration can be increased by constantly shaking the inoculated liquid cultures.Methods of Isolating Pure Culture used normally in microbiology are i) Liquid Cultures, ii) Streak Plate Method, iii) Pour Plate and Spread Plate Method, iv) Stroke Method, v) Stab method and vi) Shake Cultures.i) Liquid Cultures: These cultures can be prepared in test tubes, flasks or bottles and can be isolated by touching with a charged loop or by adding the inoculum with pipettes or syringes. Liquid cultures can be employed when concentration of the bacteria in the inoculum is expected to be small. They are preferable for inoculum containing antibiotics and other antimicrobial substances that are rendered ineffective by dilution in the media. They are also preferred when large yields are desired. The major disadvantage is that it does not provide a pure culture from mixed inoculum.

ii) Streak Plate Method: This method is routinely employed for isolation of pure culture from microbial mixture or clinical sample. The microbial mixture or sample is transferred to the edge of an agar plate with inoculating loop or swab and then streaked out over the surface in one of several patterns. At some point in the process, single cells drop from the loop as it is rubbed along the agar surface and develop into separate colonies Picture No:1- Streak Plate Method.

iii) Pour Plate and Spread Plate Method: In both of these methods the mixed culture is first diluted to provide only a few cells per milliliter before being used to inoculate media. A series of dilution is made before hand so that at least one the dilutions will contain spare concentration of cells.Picture No:2- Pour Plate and Spread Plate Methods

9

In pour –plate culture method, the mixture is diluted directly in tubes of liquid cooled agar medium. The medium is maintained in a liquid state at a temperature of 45ºC to allow through distribution of inoculum. The inoculated medium is dispensed into Petri dishes, allowed to solidify and then incubated.The disadvantages are that some of the organisms are trapped beneath the surface of the medium and there for both surface and subsurface colonies develop. The subsurface colonies can be transferred to fresh medium by digging them out of the agar with a sterile instrument. Another disadvantage is that it can be used to isolated psychrophiles as they can not tolerate the temperature of 45ºC.

In the spread plate method the mixture culture is not diluted in the culture medium; instead, it is diluted in a series of tubes containing a sterile liquid, usually water or physiological saline. A sample is removed from each tube, placed on to the surface of agar plate and spread evenly over the surface by means of a sterile bent glass rod. On at least one plate, well separated colonies will appear. In this method, only surface colonies will develop.Both of these methods are used to determine number of bacteria present in a specimen.iv) Stroke Method: This is made in tubes containing agar slopes (Slant) and is used for providing a pure growth of bacterium for slide many diagnostic tests.v) Stab method: These cultures are prepared by puncturing with a charged long straight wire in a suitable medium. The medium is allowed to set in a tube in the upright position providing a flat surface at the top of the medium.They are employed for gelatin liquefaction and oxygen requirements of bacteria under study. vi) Shake Cultures: About 10-15ml of the medium is taken in the test tubes, melted and cooled to about 40-50ºC . The inoculum is then added to the molten agar, mixed well and is allowed to set. After incubation oxygen requirements of bacteria may be studied. Obligate anaerobic organism grows only at the depths of the tube whereas facultative anaerobes grow at the surface as well as in the depths. Strict aerobes grow only at the surface of the medium.Anaerobic Culture Methods

10

A number of methods are used for culturing anaerobes. The principle is to exclude oxygen, production of vacuum condition, displacement of oxygen.i) Displacement of Oxygen: Displacement of oxygen with gases such as H2,N2,He and CO2 is sometimes employed but anaerobic conditions is seldom complete by this method alone.ii) Candle Jar Method: A popular and ineffective method is the candle jar. In this inoculated plates are placed inside a larger air tight container and a lighted candle is kept in the jar before the lid is sealed. The burning candle is expected to use up all the oxygen but some CO2 is always left behind. The candle jar provides CO2 which stimulates the growth of most bacteria.

Picture No:3-Candle JarIII. Pre-reduced Media: During preparation, cultured media is boiled for several minutes to drive off most of the dissolved oxygen. A reducing agent e.g., cystein is added to further lower the oxygen content. Oxygen –free N2 is bubbled through the medium to keep it anaerobic. The medium is then dispensed into tubes which are being flushed with oxygen-N2, stopperedd tightly and sterilized by autoclaving. Such tubes can be stored for many months before being used. During inoculation, the tubes are continuously flushed with oxygen free CO2 by means of a cannula,

restoppered and incubated.IV. Anaerobic Chamber: This refers to a plastic anaerobic glove box that contains an atmosphere of

H2, CO2 and N2. Culture media are placed within the chamber by means of an air lock which can be evacuated and refilled with N2. From the air lock the media are placed within the main chamber. Any O2 in the media is slowly removed by reaction with H2, forming water; this reaction is aided by a palladium catalyst. After being rendered oxygen –free, the media are inoculated within the chambers ( by means of glove ports) and incubated.

Picture No: 4-Anaerobic Chamber

V. Gas-Pak Jar: In this procedure environment is made anaerobic by using hydrogen and palladium catalyst to remove2 through formation of water. The reducing agent in anaerobic agar also removes Oxygen.

Picture No:5-Gas-Pak Jar

VI. Plastic Bags or Pouches: Picture No:6 - Plastic Bags for anaerobic CultureThese make convenient containers when only a few

11

samples are to be incubated anaerobically. These have a catalyst and calcium carbonate to produce an anaerobic carbon dioxide rich reagent atmosphere. A special solution is added to the pouch’s reagent compartment; Petri dishes or other containers placed in the pouch; it then is clamped shut and placed in incubator.

Maintenance and Preservation of Pure CulturesIt is very important to maintain a large collection of strains, referred to as a Stock- Culture Collection. These are required for laboratories for research and regular work. Most major biological companies maintain large cultures. These strains are used for screening of new, potentially effective chemotherapeutic agents; as assay tools for vitamins and amino acids; agents for the production of vaccines, antisera, antitumor agents, enzymes and organic chemicals; and as reference culture that are cite4d in company patents.

Methods of Maintenance and PreservationThere are various methods to maintain strains alive and uncontaminated and to prevent any change in their characteristics.i) Periodic Transfer to Fresh Media: Strains can be maintained by periodically preparing a fresh stock culture from the previous stock culture. The culture medium, the storage temperature, and the time interval at which the transfer are made vary with the species and must be ascertained before hand.The temperature and the type of medium should support a slow rather than a rapid rate of growth so that the time interval between transfers can be as long as possible. The disadvantage of this method is that it can not prevent the development of variants and mutants.ii) Overlaying Culture with Mineral Oil: This involves covering the growth on an agar slant with sterile mineral oil. The oil must cover the slant completely; to ensure this, the oil should be about ½ in above the tip of the slanted surface. Advantage include the possibility of removing some culture under oil with a transfer needle for subculture and still able to preserve the original culture. But changes in characteristic can still occur.

iii) Preservation by Lyophilzation (Freeze-Drying): Freeze-drying cab be used to preserve many culture that would be killed by ordinary drying. In this

process a dense cell suspension is placed in small vials and frozen at -60 to 78C. The vials are then connected to a high –vacuum line. The ice present in the frozen suspension sublimes under the vacuum i.e; evaporates without first going through a liquid water phase. This results in dehydration of the bacteria with a minimum of damage to delicate cell structures. The vials are then sealed off under vacuum and

12

stored in refrigerator. Advantages include that a))many species of bacteria preserved by this method have remained viable and unchanged in their characteristics for more than 30 years, b)only minimum storage space is required and c) small vials can be sent conveniently through mail.Picture No:7- Lyophilzation ProcessLyophilized cultures are revived by opening the vials, adding liquid medium and transferring the dehydrated culture to a suitable growth medium.iv) Storage at Low Temperatures: In this procedure cells are prepared as a dense suspension in a medium containing a cryoprotective agent such as glycerol or dimethyl sulfoxide(DMSO), which prevents cell damage due to ice crystal formation during the subsequent steps. The cell suspension is sealed in small ampoules or vials and then frozen at a controlled rate to -150 C. The ampoule or vials then stored in a liquid nitrogen refrigerator either by immersion in the liquid nitrogen (-196 C) or by storage in the gas phase above the liquid nitrogen (-150C). The liquid nitrogen method is advantages for many species which can not be preserved by lyophilization. The cultures remain viable for 10-30 years without undergoing changes. Disadvantage is that the method is expensive, since the liquid nitrogen in the refrigerators must be replenished at regular intervals to replace the loss due to evaporation.

Cultural Characteristics (Morphology) of Microorganisms.When grown on a variety of media, microorganisms will exhibit differences in the microscopic appearance of their growth. These differences, called cultural characteristics, are used as the basis for separating microorganisms into taxonomic groups.The cultural characteristics for all known microorganisms are contained in Bergey’s Manual of Systemic Bacteriology . They are determined by culturing the organisms on nutrient agar slants, and plates, in nutrient broth and in nutrient gelatin. The patterns of growth to be considered are described as under:Characteristics on Nutrient Agar: This demonstrates well isolated colonies and are evaluated in the following manner:1.Size: Pinpoint, small, moderate or large

13

Picture No:8-Cultural Characters of Bacteria2. Form: The shape of colony is described as:a) Circular-Unbroken, peripheral edge, b) Irregular: Indented, peripheral edge, c) Rhizoid: Root like, spreading growth.3.Elevation:The degree to which colony growth is raised on the agar surface is described as:a) Flat: Elevation not discernibleb) Raised: Slightly elevatedc) Convex: Dome-shaped elevationd)Umbonate: Raised with elevated convex central region.4. Margin:The appearance of the outer edge of the colony is described as :a) Entire: Shapely defined, even, b) Lobate: marked indentations, c) Undulate: Wavy indentations,d) Serrate (Erose): Toothlike appearance, e) Filamentous: Threadlike, spreading edge.5. Pigmentation: Color of Colony

Characteristics on Nutrient Agar Slant:a) Abundance of growth: The amount of growth is designated as none, slight, moderate or large.b)Pigmentation: Chromogenic microorganisms may produce intracellular pigments that are responsible for coloration of the organisms. Other organisms produce extracelluar soluble pigments that are excreted into medium and that also a color.c) Optical Characteristics: It is described as opaque, transparent and translucent.d) Form:The appearance of the single-line strak of growth on the agar surface is designated as:

a) Filiform: Continuous ,threadlike growth with smooth edges.b) Echinulate : Continuous thread like growth with irregular edges.c) Beaded: Nonconfluent to semiconfluent colonies.d) Effuse: Thin, spreading growth.e) Arborescent: Thread like growthf) Rhizoid: Rootlike growth